Dominant Drug Targets Suppress the Emergence of Antiviral Resistance

RESEARCH ARTICLE elifesciences.org Dominant drug targets suppress the emergence of antiviral resistance Elizabeth J Tanner1, Hong-mei Liu2, M Steven Oberste2, Mark Pallansch2, Marc S Collett3, Karla Kirkegaard1* 1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States; 2Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, United States; 3ViroDefense, Inc., Rockville, United States Abstract The emergence of drug resistance can defeat the successful treatment of pathogens that display high mutation rates, as exemplified by RNA viruses. Here we detail a new paradigm in which a single compound directed against a ‘dominant drug target’ suppresses the emergence of naturally occurring drug-resistant variants in mice and cultured cells. All new drug-resistant viruses arise during intracellular replication and initially express their phenotypes in the presence of drug-susceptible genomes. For the targets of most anti-viral compounds, the presence of these drug-susceptible viral genomes does not prevent the selection of drug resistance. Here we show that, for an inhibitor of the function of oligomeric capsid proteins of poliovirus, the expression of drug-susceptible genomes causes chimeric oligomers to form, thus rendering the drug-susceptible genomes dominant. The use of dominant drug targets should suppress drug resistance whenever multiple genomes arise in the same cell and express products in a common milieu. DOI: 10.7554/eLife.03830.001 Introduction Drug-resistant variants of positive-strand RNA viruses are generated due to error-prone replication of *For correspondence: karlak@ parental, drug-susceptible genomes. Even if individual host cells are infected with only a single stanford.edu virus each, error rates of 1 × 10−4 per nucleotide or higher ensure that each cell will experience a mixed Competing interests: See page 14 infection as variant progeny genomes arise within it (Holland et al., 1989). The new variants act as templates for both continued genome synthesis and translation of new proteins. What happens in the Funding: See page 14 face of a new selective pressure, such as the addition of an antiviral drug? In most cases, antiviral treat- Received: 28 June 2014 ments prevent the growth of drug-susceptible viral genomes, but any drug-resistant variants in the Accepted: 01 November 2014 same cell continue to amplify according to their fitness (Figure 1A, top). Published: 03 November 2014 In contrast, the dominant drug target approach presented here exploits the potential interactions between the products of drug-resistant and drug-susceptible genomes within the same cell that Reviewing editor: Wenhui Li, National Institute of Biological render the drug-susceptible phenotype dominant (Crowder and Kirkegaard, 2005). The most intui- Sciences, Beijing, China tive scenario for such dominance involves highly oligomeric assemblages such as viral capsids, because drug-resistant subunits will assemble with drug-susceptible ones, and the chimeric structure is likely to This is an open-access article, be inhibited by the drug (Figure 1A, bottom). This situation is akin to a dominant–negative interaction free of all copyright, and may be and examples of such dominance by vector-mediated expression of mutant proteins are abundant. freely reproduced, distributed, Expression of a non-functional version of the oligomeric Tat protein of HIV drastically reduces viral transmitted, modified, built upon, or otherwise used by yield (Meredith et al., 2009). Similarly, co-expression of non-functional NS5A protein of hepatitis C anyone for any lawful purpose. virus inhibits replication of genomes that encode functional NS5A (Graziani and Paonessa, 2004). In The work is made available under poliovirus, viral genomes with mutations in several proteins, including capsid proteins and the RNA- the Creative Commons CC0 dependent RNA polymerase, interfere with the amplification of co-transfected wild-type viral genomes public domain dedication. (Crowder and Kirkegaard, 2005). Tanner et al. eLife 2014;3:e03830. DOI: 10.7554/eLife.03830 1 of 16 Research article Genes and chromosomes | Human biology and medicine eLife digest Treating a viral infection with a drug sometimes has an unwanted side effect— the virus quickly becomes resistant to the drug. Viruses whose genetic information is encoded in molecules of RNA mutate faster than DNA viruses and are particularly good at developing resistance to drugs. This is because the process of copying the RNA is prone to errors, and by chance some of these errors, or mutations, may allow the virus to resist the drug's effects. Treating viral infections with most drugs destroys the viruses that are susceptible to the drug and inadvertently ‘selects’ for viruses that are resistant to the drug's effects. These drug-resistant viruses are harder to treat and often require physicians to switch between different drugs. Sometimes these new drug-resistant viruses spread and these new infections cannot be treated with drugs that would have worked in the past. So far, the best strategy to prevent drug-resistant viruses from growing in patients is to use multiple drugs, such as the life-saving treatments for HIV infection. However, for many viral infections—such as those that cause the common cold, dengue fever, Ebola, and polio—no drugs are yet available to treat infected people. Moreover, there are concerns that, if a new drug is used on its own, the viruses will quickly develop resistance to the drug and render it ineffective. Tanner et al. now show that an antiviral drug that interferes with the formation of the outer layer (or capsid) of the poliovirus inhibits the emergence of drug resistance. The drug, called V-073, is currently being tested as a treatment for poliovirus and will be useful in the worldwide eradication effort. Tanner et al. show that treating poliovirus-infected mice with V-073 does not select for drug-resistant strains of the virus—and provide evidence that this occurs because the drug targets an assemblage of proteins. The poliovirus capsid is assembled from a mix of proteins from different naturally occurring strains of the virus within the infected cell. A new strain of virus is always ‘born’ into a cell that is already infected by other viruses, which could be thought of as its parents, cousins and siblings. A new drug-resistant virus will therefore be forced to mix its capsid proteins with those of its ‘family’ members, who are all drug-sensitive. These hybrid capsids will remain vulnerable to the drug—and in this way, the resistant strains do not become the dominant form of the virus. Tanner et al. also discovered a way to screen for drugs that have a similar resistance-blocking effect. These drugs would target capsids, or other viral structures made up of a mix of proteins from different virus strains. Such drugs might be useful against other viruses including the ones that cause the common cold, hepatitis C, or dengue fever. DOI: 10.7554/eLife.03830.002 The idea of dominant drug targeting differs from conventional dominant–negative interaction experiments in two ways. First, the dominant products are encoded by the wild-type, drug-susceptible genome that initiated the infection as well as its drug-susceptible progeny. The second unique feature is that the mixed infection of drug-susceptible and drug-resistant viruses does not result from co- infection or co-transfection. Instead, the intracellular diversity arises from the error-prone replication of the viruses themselves, even if the infection in any individual cell is initiated by a single viral genome. Here, we show that V-073, an inhibitor of viral capsid function that is currently in development for use in the poliovirus eradication campaign (Buontempo et al., 1997; Collett et al., 2008; Oberste et al., 2009), fulfills the expectations of dominant drug targeting, dramatically suppressing the growth of drug resistant viruses in murine infections and in cell culture via the formation of mixed assemblages. Results Growth of wild-type virus during treatment with guanidine allows selection of guanidine-resistant viruses in mice The active sites of enzymes are often considered to be promising drug targets, due to a wealth of biochemical analyses and thousands of precedents. For poliovirus, one such target is 2C protein, a membrane-associated NTPase that is required for viral genome replication. The NTPase activity of protein 2C is inhibited by low concentrations of guanidine in cell culture and in solution (Pfister and Wimmer, 1999). We investigated the effectiveness of guanidine in inhibiting viral growth in mice and Tanner et al. eLife 2014;3:e03830. DOI: 10.7554/eLife.03830 2 of 16 Research article Genes and chromosomes | Human biology and medicine Figure 1. Emergence of drug-resistant variants in non-dominant and dominant drug targets in mice. (A) Representation of drug-resistant viral amplification when a non-dominant drug target is inhibited (top) but its inhibition by drug-susceptible variants when a dominant drug target, such as an oligomeric structure, is inhibited (bottom). See text for discussion. (B–K) Tnfr1+/+ (B, C) or Tnfr1−/− (d-k) PVR-expressing mice were infected with 1 × 107 PFU Mahoney type 1 poliovirus by intramuscular inoculation and treated with 76 mg/kg/day guanidine (B–E, green symbols) or 10 mg/kg/day V-073 (1-[(2-chloro-4-methoxyphenoxy)methyl]-4-[(2,6-dichlorophenoxy)methyl]benzene) (F–K, orange symbols). Inoculated muscles were harvested at times indicated. Total viral yields (PFU/mg tissue) in muscle samples of mice treated with guanidine (B, D), V-073 (F, H, J) or vehicles (black) are shown. Fold changes in the frequency of drug-resistant variants in mice treated with guanidine (C, E) or V-073 (G, I, K) are shown. Fold change calculated by dividing sample value by mean value of control mice. p-values: 0.02 (B), 0.003 (C), 0.0002 (D) 0.02 (E), and 0.01 (F, H, J combined).

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